Figure 3. Regulated genes in transgenic mice and validation of Ctgf as a target.

(A): qRT-PCR using limb RNA (E12.5-E14.5) from wildtype (Wt) and transgenic littermates (Tg) (N = 8–10 for each stage). Measurements were carried out individually, in duplicates, and normalized to Adam9 and Sdha. Relative normalized values are presented on the y-axis. Significances are indicated in each diagram by asterisks (*: p≤0.05, **: p≤0.01, ***: p≤0.001). Variations are indicated by the standard deviation (SD). In 7/8 candidates an upregulation was confirmed as significant in at least one embryonic stage. (B): nCounter analysis of CTGF and SHOX expression in NHDF and U2OS cells after transient transfections of SHOX and p.Y141D. CTGF is significantly downregulated in NHDF cells, whereas it is significantly upregulated in U2OS cells. Values on y-axis represent absolute counts of mRNA, normalized to ADAM9, HPRT1 and SDHA. Significancies are indicated by asterisks. (C): In situ hybridization using a Ctgf antisense riboprobe on embryonic limbs from wildtype and SHOX-transgenic littermates (N = 8) at stage E12.5. In transgenic embryos, enhanced and distalized expression of Ctgf was detected in the middle part of the developing limbs.